The present invention relates to antisense oligonucleotides, and more particularly to chimeric antisense oligonucleotides which exhibit high resistance to endo- and exonucleases, high sequence specificity, and the ability to activate RNAse H, as evidenced by efficient and long-lasting knockout of target mRNA. Also provided are formulations of the oligonucleotides with carrier molecules which provide efficient transfection into cells.
The use of antisense oligonucleotides to specifically inhibit the function of targeted genes has been the subject of extensive research, due to its promise in selective antiviral and anticancer therapy. Many studies have been directed to the design of oligonucleotide analogs having an optimal combination of properties, including stability (i.e. resistance to cellular nucleases), cellular uptake, DNA/RNA binding affinity and specificity, and efficiency of inhibition. Because the phosphodiester linkages of native nucleic acids are degraded by endo- and exonucleases, many early studies were directed to designing nuclease-resistant analogs. Phosphorothioates are one such class of compounds, which are relatively stable in vivo and retain the ability to activate RNAse H, the primary mechanism by which antisense oligonucleotides deactivate target RNA. However, the use of phosphorothioates presents several disadvantages, including a high level of non-specific binding to other cellular components, often leading to unwanted side effects, and reduced binding affinity for RNA.
Oligomeric ribonucleotides substituted at the 2xe2x80x2-oxygen show high RNA binding affinities and, in comparison to the unsubstituted ribonucleotides, reduced sensitivity to endogenous nucleases. Although 2xe2x80x2-O-methyl substituted ribonucleotides provide greater binding affinity than those having larger substituents (e.g. ethyl, propyl, pentyl, allyl), the larger substituents are reported to confer greater exonuclease resistance (see, for example, Monia et al., J. Biol. Chem. 271(24): 14533, 1996). Arrow et al. (U.S. Patent No. 5,849,902) stated that xe2x80x9c2xe2x80x2-O-methyl bases with phosphodiester linkages are degraded by exonucleases and so are not suitable for use in cell or therapeutic applications of antisense.xe2x80x9d Phosphorothioate and phosphotriester linkages were recommended by the latter group as having greater stability, even though they presented the disadvantages of reduced binding affinity, more difficult synthesis (phosphotriester) and/or greater toxicity (phosphorothioate).
Therefore, there is still a need for antisense oligonucleotide compositions with optimal combinations of antisense activity, target binding affinity, biocompatibility, and stability.
The present invention includes, in one aspect, a chimeric oligonucleotide having the formula 5xe2x80x2-W-X1-Y-X2-Z-3xe2x80x2, where W represents a 5xe2x80x2-O-alkyl nucleotide, such as a 5xe2x80x2-O-alkyl thymidine; each of X1 and X2 represents a block of seven to twelve phosphodiester-linked 2xe2x80x2-O-alkyl ribonucleotides; Y represents a block of five to twelve phosphorothioate-linked deoxyribonucleotides; and Z represents a blocking group effective to block nuclease activity at the 3xe2x80x2 end of the oligonucleotide. In one embodiment, Z is a 3-to-3xe2x80x2 linked nucleotide. In further embodiments, the alkyl groups of the 5xe2x80x2-O-alkyl nucleotide and/or the 2xe2x80x2-O-alkyl ribonucleotides are methyl groups. In still further embodiments, groups W and/or Z are linked to X1 and X2, respectively, via phosphodiester linkages, phosphotriester, phosphorothioate, or phosphoramidate linkages. Preferably, W is linked via a phosphodiester or phosphorothioate linkage, and Z is linked via a relatively nuclease-resistant linkage; i.e. a phosphotriester, phosphorothioate, or phosphoramidate linkage.
In specific embodiments, the segment X1-Y-X2 of the chimeric oligonucleotide has a sequence represented by any of SEQ ID NOs: 1-24 disclosed herein.
In another aspect, the invention provides a therapeutic composition which comprises an oligonucleotide as described above in a pharmaceutically acceptable vehicle. In preferred embodiments, the vehicle includes a lipid-cationic peptoid conjugate or xe2x80x9clipitoidxe2x80x9d. One class of lipid-cationic peptoid conjugates includes compounds of the formula:
L-linker-[N(CH2CH2NH2)CH2(Cxe2x95x90O)xe2x80x94N(CH2CH2R)CH2(Cxe2x95x90O)xe2x80x94N(CH2CH2R)CH2(Cxe2x95x90O)]3xe2x80x94NH2,
where the lipid group L is a fatty acid-derived group, such as a phospholipid group (i.e. ROOCCH2CH(COOR)CH2OP(O)2Oxe2x80x94), having fatty alkyl or alkenyl chains between about 8 and 24 carbon atoms in length, or a steroid-derived group, such as a cholesteryl group, and the portion of the molecule to the right of the linker is the peptoid segment. In the peptoid segment, R is selected from alkyl (branched or unbranched), aminoalkyl, and aralkyl. As used herein, xe2x80x9caralkylxe2x80x9d refers to an alkyl, preferably lower alkyl, substituent which is further substituted with an aryl group; one example is a benzyl group. xe2x80x9cArylxe2x80x9d refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., benzene) or two condensed rings (e.g., naphthyl). This term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as furyl, pyrrole, pyridyl, and indole. By xe2x80x9csubstitutedxe2x80x9d is meant that one or more ring hydrogens in the aryl group is replaced with a substituent, preferably selected from a halide, a lower alkyl or lower alkoxy group, halomethyl, or haloethyl.
In specific embodiments, R is isopropyl or 4-methoxyphenyl. A single lipitoid may include different groups R, or they may be the same within the molecule.
The linker may be a direct bond, or it may be a substantially linear linking group, such as an oligopeptide or an alkyl chain, of any effective length. The linker may also be an alkyl chain having one or more heteroatom-containing linkages, selected from the group consisting of ester, amide, carbonate, carbamate, disulfide, peptide, and ether, at either terminus of the chain or intervening between alkyl bonds. In selected embodiments, the linker is from 2 to about 30 atoms, or from 3 to about 15 atoms, in length.
In another aspect, the invention provides a method of inhibiting expression of a target gene in a subject, which comprises administering to the subject, in a pharmaceutically acceptable vehicle, an amount of a chimeric oligonucleotide effective to specifically hybridize to all or part of a selected target nucleic acid sequence derived from the gene, where the chimeric oligonucleotide has a structure as described above. In one embodiment, the target nucleic acid sequence is a mRNA derived from the target gene. In specific embodiments, the segment X1-Y-X2 of the chimeric oligonucleotide has a sequence represented by any of SEQ ID NOs: 1-24 disclosed herein. In further embodiments, the vehicle includes a lipid-cationic peptoid conjugate such as described above.